Viral encephalitis is rising as a cause for alarm, with the recent emergence of a series of new agents of serious CNS infection, including several zoonotic viruses that cause lethal encephalitis in humans. One virus that has long been a human pathogen remains at the top of the list: measles. Measles virus (MV) causes disease worldwide despite efforts towards eradication by vaccine, largely because it is spread so readily between people. While the disease is generally self-limited, several serious complications involve the CNS and lead to sequelae or death. These CNS manifestations may occur early after infection, in the case of acute encephalomyelitis, or years after infection, as a result of viral persistence in subacute sclerosing panencephalitis. The third form of MV-induced CNS disease, progressive infectious encephalitis, occurs 1 to 6 months following measles infection, and is problematic in an increasing population of immunocompromised patients. There is no specific therapy for acute or persistent CNS complications of measles. We propose to apply the results of our fundamental research to the development of a new antiviral strategy for measles CNS infection. The proposal is based on our recent discovery that attachment of a cholesterol group to a peptide fusion inhibitor yields 3 major advantages: (1) increased potency, (2) localization of the peptide with the virus at the site of fusion activation, and (3) CNS penetration. We showed that cholesterol- tagged peptides are highly effective against measles virus in vitro, and we propose to assess their potential to treat measles infection of the CNS in a transgenic murine encephalitis model expressing the MV receptor human CD150 (SLAM). By combining sequence optimization with cholesterol tagging, we propose to develop highly effective peptide fusion antivirals that inhibit MV in vitro, and to test their therapeutic potential in a relevant animal model of MV encephalitis. We will pursue preclinical development of our lead therapeutic candidate by: Aim 1. Lead optimization of MV fusion inhibitor peptides targeted to the plasma membrane, using (a) biophysical analysis-guided sequence optimization, and (b) membrane targeting. Aim 2. Effectiveness of the fusion inhibitors to protect from measles encephalitis induced by wild type MV in a transgenic murine model. We will assess (a) bioavailability of the targeted peptides, and (b) efficacy in challenge experiments in a CD150(SLAM) transgenic mouse model. We will thereby obtain proof of principle for efficacy, allowing us to select peptides for advancement that have the most in vivo potential.